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Apparently, there's no way to extricate the parasite without eliminating the host. Fancy way of saying we gotta kill the fucking kid. And now they're asking for my go ahead. The tests just keep getting harder and harder, don't they? I'm so tired. I'm exhausted and I just want this to end... so be it. [...] Oh I miss you Anna. Your daughter will be with you soon.

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As a Firefly, Marlene fought against the totalitarian rule of the military within the Quarantine Zones to restore a form of functional government. The Fireflies have been successful in overthrowing military rule in several zones, namely Salt Lake City but their efforts have not always achieved the desired results, as in Pittsburgh. Despite her and her organization's attempts to restore order, their numbers have dwindled as the military cracked down and killed members found within the zones. In addition to all this, Marlene had her organization keep a close eye on Ellie as part of her promise to Anna.

She was once close with Joel's brother, Tommy, when he was a member of the Fireflies. After a while, however, Tommy gave up on the cause and decided to leave. Before he left, he told Marlene that if she ever needed anything, she could count on Joel to help her.

After retreating from a losing firefight with the Boston military, Marlene arrived in time to save Ellie and Riley from the infected in an abandoned mall outside the zone. She had the two girls gagged and tied up before taking them to a smugglers' tunnel. Coming back from finding some bread and other things, Marlene later freed the girls of their bindings. Riley immediately requested that she join the Fireflies but Marlene denied, reasoning that too many men had died for the cause. Their argument was cut short when hostile smugglers arrived and opened fire on the group.

Once the smugglers were killed, Riley persisted in joining and dying for the cause if necessary, prompting Marlene to point a gun at her. Thinking that she would shoot, Ellie grabbed Trevor's gun and pointed it at Marlene to make her drop hers. Marlene did so but stated that she never intended to kill Riley. She then revealed that she was friends with Ellie's mother Anna and gives Ellie a switchblade as well as a letter. Marlene then directed the two girls back to the military boarding school in the quarantine zone.

Sometime afterwards, Marlene had the Fireflies lure Riley to her, who was still looking to join the Fireflies. Her men cornered Riley in an alley when she tailed Trevor, and took the girl to Marlene, only stating "what took you so long?" and revealed that she wanted to test her resolve when they last met. As part of Riley's initiation into the Fireflies, Marlene had the girl kill an Infected. When Riley asked whether Ellie could join the organization, Marlene told her that she wanted Ellie to stay safe at the military school and forbade Riley to see her again. Understanding Riley and Ellie's relationship, Marlene decided to send Riley away to another city further away from their unit. Riley argued heavily against such but Marlene ignored her pleas, planning the move the day after Riley sneaked back to see Ellie.

Six weeks later Ellie and Riley were bitten at the same abandoned mall. Marlene took Ellie back under her wing, almost shooting her when she found out she'd been bitten. When Ellie failed to succumb to the CBI as her friend Riley did, Marlene realized that she was immune. This is key to their cause--finding a vaccine.

Looking to keep up the morale of the Fireflies, Marlene made a deal with a smuggler named Robert and purchased a cache of guns. She went to close the deal and, during events unseen, she was badly injured. When she finally located Robert, Tess and Joel had killed him. Tess demanded her guns from Marlene, who refused to give them up at first. Marlene decided to concede when she asked them to smuggle something out of the city for her.[3] Tess agreed on the basis that she was allowed to see the weapons before committing entirely.[4]

Marlene guided them through the docks and around the military patrols still searching for the Fireflies that attacked the checkpoint within the city. She explained that the Fireflies attempted to leave the city, but the military "riled them up". As a result, the Fireflies have fought to defend themselves.

Marlene successfully led them back to her hideout as she collapsed. Assuming Marlene was in danger, Ellie tried to attack Joel from behind with a switchblade, but Tess stopped her. Marlene assured her friend she would recover, but she could come with her. Ellie wanted to stay with her, but Marlene reminded her that this was their only chance to get her out of the city. Marlene asked Joel to watch over Ellie during their journey to the capitol building, an idea Ellie objected to. Marlene assured her that she could trust Joel. Once Ellie was safe with Joel, Marlene took Tess to verify the weapons.[5]

Once Marlene recovered from her injuries, she and the rest of her surviving men left Boston and moved to join up with the rest of the Fireflies in Salt Lake City. Along the way, she and her men were attacked by the Infected losing two of their members, leading to the group entering a dismal phase. However, they soon brightened up, much to Marlene's happiness. By March 2034, Marlene barely made it to the city with the rest of her men intact. They reached the hospital, the group and Marlene entering better spirits at bring reunited with friends they hadn't seen in as long as ten years. She soon learned that the Fireflies that were supposed to meet Ellie and the smugglers at the Capitol Building were already killed. With no way of knowing where they are, coupled with the losses she endured during her trek with her men across the country, Marlene sank into depression over her capabilities as a leader, even being unable to eat. Just a month later, April 25th, Marlene even began to doubt her cause as a Firefly, feeling a need to run away from her team though she did not go through with it.[6]

On April 28, 2034, Firefly scouts reported that they spotted an older man and a young girl in the tunnels, who turned out to be Joel and Ellie. Joel was knocked unconscious by a Firefly while Ellie, having nearly drowned, was taken to the surgery room to be examined. Initially overjoyed to see them, the doctors told Marlene that the unique mutation of CBI within Ellie made it possible for them to reverse-engineer a vaccine. However, the doctors could not operate on her friend’s brain without killing her. The doctors asked for her “permission” to go ahead with the procedure, a formality Marlene assumed was done merely on principal as she did not think she could stop the operation regardless. She decided to make the choice and allowed them to proceed. The decision was painstaking on her. She recorded two ruminations to Anna over the choice she was about to make, but remained firm that it needed to be done.[7]

Marlene thanked Joel for looking after Ellie, grateful they both survived the ordeal to get to the hospital. When she told him of Ellie’s situation and the possibility of a vaccine being produced, he objected to the idea. Marlene, sympathetic to his turmoil, told him that Ellie’s death was necessary and far more important than anything either of them may feel for her as surrogate parents. She tols the Firefly with her, Ethan, to lead him out but only shoot him should he attempt to stop the operation. Earlier, other Fireflies suggested to Marlene that she just kill Joel, but she refused to kill the one person who understood what she was going through.[8]

Her decision to keep him alive allowed Joel, having killed Ethan, to stop the operation and force Ellie from the doctors. Marlene cornered Joel in the parking lot of the hospital to dissuade him at gunpoint. She tried to intimidate him with terrible scenarios that could happen to Ellie; death at the hands of Clickers, sexual assault, and/or murder committed by thugs.

Marlene told him that Ellie would want to follow through with the surgery and that he could still do the right thing by handing her over. She lowered her gun and raised her hands in a show of peace, but Joel shot her in the side and placed the unconscious Ellie inside a car. Bleeding out, Marlene begged Joel to let her live. However, he stated that she would only pursue Ellie to get her back, so he shot Marlene point blank in the face.[9] After driving away, Joel lied to Ellie that Marlene and the Fireflies gave up looking for a cure, so that she could get over her survivor's guilt.[10]

It is implied that Marlene and Tess knew each other before the events of the game; Marlene going so far as to call Tess by name. Whether their relationship was personal or through mutual knowledge of their respective notoriety is unknown. Tess cared about her to some degree, asking if she was okay when showing signs of pain due to her bullet wound. However, Marlene was viewed as untrustworthy by Tess, by how she refused to do any smuggling until she saw the merchandise she promised, yet went with her to check it out, trusting that the Firefly would not harm her. Their relationship appeared to be one of business, the two only interacting with each with the interest in using the other for personal gain; Marlene by giving guns to Tess, and Tess by escorting Ellie to safety for Marlene.

Joel knew Marlene as the leader of the Fireflies ever since his brother Tommy joined the militia group. Before leaving, Tommy told her that if she was ever in a jam she could rely on Joel for help. Joel is quite cynical of the Fireflies' promises of hope, which was flowed on to Marlene.

When Joel did finally deliver Ellie to the Fireflies, Marlene compared his bond with Ellie to her own and understood that, as Ellie's surrogate parents, the decision to kill her to develop a vaccine was hard on her and Joel. However, Marlene reluctantly decided to kill her while Joel attempted to save Ellie from that fate, demonstrating how different the pair's philosophies were; Marlene had always been about saving humanity no matter the cost despite how personal it became for her, while Joel had always been about himself, learning to detest humanity rather than want to save it. The clash of these juxtaposed philosophies culminated in Joel killing Marlene in fear that she would come after Ellie.

Marlene was described by Ellie as "just a friend, I guess." Marlene treated Ellie as her surrogate daughter, following her friend Anna's dying wish. Despite the affection she gained for Ellie, protecting her from the military and later believing she was immune, Marlene was still willing to sacrifice Ellie in order to find a vaccine. She pleaded her guilt to Joel, remarking that what he's been through was "nothing to what [she's] been through".

The pair were close enough that Ellie didn't want to part from her with Joel and Tess and felt a need to protect her when she saw Joel enter the room with a visibly wounded Marlene.

Although not much was displayed of her relationship with him, Marlene respected Ethan, the man being an ally of hers due to their shared beliefs. Although she ordered him around, Ethan doesn't mind, he was merely following her commands as a loyal Firefly. Marlene put faith in him to escort Joel out of the hospital though this overconfidence in Ethan enabled Joel to escape.

Marlene became close with Tommy during his time as a Firefly. They were close enough that he told her about his brother Joel and even recommended him if she "was ever stuck in a jam". The two rarely saw each other after he left the Fireflies, but she still believed "he was a good man".

"Apparently, there's no way to extricate the parasite without eliminating the host. Fancy way of saying we gotta kill the fucking kid. And now they're asking for my go ahead. The tests just keep getting harder and harder, don't they? I'm so tired. I'm exhausted and I just want this to end … so be it.

"Hey Anna... It's been awhile since we spoke. I uh... I just gave the go ahead to proceed with the surgery. I really doubt I had much of a choice, asking me was more of a formality. I need you to know that I've kept my promise all these years... despite everything that I was in charge of, I looked after her. I would've done anything for her, and at times... Here's a chance to save us... all of us. This is what we were after... what you were after. They asked me to kill the smuggler. I'm not about to kill the one man in this facility that might understand the weight of this choice. Maybe he can forgive me. Oh I miss you Anna. Your daughter will be with you soon."

Tess: "Look, let's go find a Firefly."

"You won't have to look very far."

"I don't give a damn about ration cards. I need something smuggled out of the city."

"Been planning on leaving the city, but they need a scapegoat. They've been trying to rile us up."

"We can sneak by them, even though I know that's not your style."

"Goddammit, they got Warren. Goodbye, friend.'"

Joel: "You're recruitin' kind of young, aren't ya?"

"She's not one of mine."

"You'll follow me. You can verify the weapons. I can get patched up. But she's not crossing to that part of town."

"Welcome to the Fireflies."

"Look … you can still do the right thing here."

"I lost most my of crew crossing the country. I pretty much lost everything. And then you show up and somehow we find you just in time to save her. Maybe it was meant to be."

"Don't waste this gift, Joel."

"Because this isn't about me. Or even her. There is no other choice here."

"I'm not about to kill the one man in this facility that might understand the weight of this choice."

"You can't save her. How long before she's torn to pieces by a pack of Clickers? And that's if she hasn't been raped and murdered first."

Ellie refers to Marlene as "just a friend, I guess" even though Marlene took care of her shortly after she was born. As such, it is unusual she thinks Joel is closer to her than Marlene, possibly because Marlene sent her to a military boarding school. Given Ellie's emotionally closed-off personality, it is also possible that she may have felt uncomfortable opening up to talk about Marlene as a close friend.

Creative director Neil Druckmann remarked in the in-game commentary of the game's cutscenes he unintentionally had Marlene mirror David's belief: "everything happens for a reason"; which Marlene indirectly reiterates to Joel: "maybe it was meant to be". He remarked it likely happened due to him "thinking about it, so naturally the characters end up talking about it as you're writing."

Originally, Marlene died in the surgery room during a cinematic, but Naughty Dog nixed the idea and removed the cutscene.[11]

During her initial conception, Marlene was merely a doctor who knew of Ellie's immunity and where the people developing a cure could be found. She escorted Joel and Ellie to the outskirts only to be bitten and die of infection, replacing Tess' role (who was originally the primary antagonist). A programmer at Naughty Dog later suggested Marlene be brought back and expanded upon, eventually becoming the leader of those still finding a cure (who later developed into the Fireflies).